Yeast cells are among the cell types that have been employed as host cells in the production of heterologous proteins through recombinant DNA technology. Secretion of the desired protein from the yeast cells is generally advantageous for reasons that include facilitating the purification process, since the desired protein is recovered from the culture supernatant rather than from the complex mixture of proteins that results when yeast cells are disrupted to release intracellular proteins. Secretion also reduces the deleterious (e.g., toxic) effect that intracellular accumulation of a given foreign protein may have on the host cell.
Secreted proteins generally are initially expressed as precursors bearing an N-terminal signal or leader peptide. Signal peptides generally contain a positively charged N-terminus followed by a hydrophobic core, followed by a recognition site for an enzyme known as signal peptidase. This enzyme cleaves the signal peptide from the protein during translocation. The protein is transported from the endoplasmic reticulum to the Golgi apparatus, and then follows one of a number of routes in the secretory pathway, depending on the nature of the protein. The protein may be secreted into the culture medium or may be retained on the cell surface, for example. Certain receptors that comprise extracellular, transmembrane, and cytoplasmic domains are examples of proteins that may be retained on the cell membrane, with only the extracellular domain located outside the cell.
The leader sequences of certain secreted proteins comprise peptides that are located C-terminal to the signal peptide and are processed from the mature protein of interest subsequent to cleavage of the signal peptide. Such leaders often are referred to as prepro peptides, wherein the pre region is the signal sequence and the pro region designates the remainder of the leader. One example is the yeast .alpha.-factor leader, which contains a signal peptide (including a C-terminal signal peptidase recognition site AlaLeuAla) followed by a pro region containing a basic amino acid pair LysArg that constitutes a KEX2 protease processing site, immediately followed by a peptide GluAlaGluAla (SEQ ID No:7) at the C-terminus of the pro region. Processing of this leader involves removal of the signal peptide by signal peptidase, followed by cleavage between the Lys and Arg residues by KEX2 protease. The GluAlaGluAla (SEQ ID No:7) residues are subsequently removed by a peptidase that is the product of the STE13 gene (Julius et al., Cell 32:839, 1983). The yeast .alpha.-factor leader is described in U.S. Pat. No. 4,546,082.
Signal peptides derived from proteins naturally secreted by yeast cells have been employed in recombinant expression systems for production of heterologous proteins in yeast. The use of mammalian signal peptides in yeast expression systems also has been reported, although certain of the mammalian signal peptides were not effective in promoting secretion of heterologous proteins in yeast.
Research continues to be directed toward increasing the levels of secreted and correctly processed recombinant proteins produced in yeast expression systems. Such improved expression systems would provide advantages such as the cost savings afforded by more efficient production and purification processes, and the time savings realized by researchers when protein purification is simplified through efficient secretion of the desired protein from the cell. A need remains for alternative and improved expression systems for producing recombinant proteins in yeast. This goal may be pursued by isolating new yeast strains having advantageous properties, or by developing new expression vectors.